Inhibiting proinflammatory cytokine production

ABSTRACT

There is disclosed a method for helping to prevent miscarriages during pregnancy, comprising administering an effective amount of a fetuin polypeptide.

CROSS REFERENCE TO RELATED APPLICATION

The present patent application is a divisional of U.S. patent application Ser. No. 08/932,871 filed Sep. 18, 1997 now U.S. Pat. No. 6,011,005.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a method for prevention of miscarriages during pregnancy.

BACKGROUND OF THE INVENTION

Pregnancy has been termed "Nature's transplant" because the developing fetus, essentially a foreign tissue graft, is protected from rejection by its host, the mother (Editorial: "Nature's transplant" Lancet 1:345-346, 1974). Rejection of a transplanted allograft in an immunocompetent host is normally mediated by the macrophage-derived cytokine tumor necrosis factor (TNF) (Eason et al., Transplantation 59:300-305, 1995). Excessive production of TNF during pregnancy causes spontaneous abortion (Shaarawy et al., Acta Obstet. Gynecol. Scand. 76:205-211, 1997; and Mallmann et al., Arch. Gynecol. Obstet. 249:73-78, 1991). Recently, spermine, a ubiquitous biogenic amine present in large amounts in the amnion, has been shown to counter-regulate the immune response by inhibiting the production of TNF and other pro-inflammatory cytokines by human mononuclear cells (Zhang et al., J Exp. Med. 185:1759-1768, 1997).

Fetuin is a globular 341-amino acid protein containing 20-25% carbohydrate (by weight) and 6 internal disulfide bonds. The human fetuin sequence (also known as α2-HS glycoprotein) is provided herein as SEQ ID NO. 1 and SEQ ID NO. 2. Fetuin was first identified over 50 years ago as a major protein component of bovine fetal serum but its biological function remains unclear. Bovine fetuin is a globular 341 amino acid polypeptide with six internal disulfide bonds and three N-linked and two O-linked oligosaccharide chains. Primary amino acid sequence and the position of cysteine residues are well conserved in human, bovine, sheep, rat and mouse fetuin homologs (Dziegielewska et al., J Biol. Chem. 265:4354, 1990; Rauth et al., Eur. J Biochem. 205:321,1992; Lee et al., Proc. NatL. Acad. Sci. USA 84:4403, 1987; and Brown et al., Eur. J Biochem. 205:321, 1992). Fetuin levels in human plasma are regulated in the manner of a negative acute phase reactant (Lebreton et al., J Clin. Invest. 64:1118, 1979). IL-1 was shown to suppress fetuin transcript levels in cultured hepatocytes (Akhoundi et al., J Biol. Chem. 268:15925, 1994). Fetuin appears to be expressed in bone because transcripts have been detected in both chondrocytes and osteoblasts (Yang et al., Blood 12:7, 1991). The polypeptide α2-HS glycoprotein is a human homolog of fetuin and is secreted in high levels by adult liver into the peripheral circulation (Triffitt et al., Nature 262:226, 1976).

Human fetuin has 3 N-linked oligosaccharide chains (attached to the amine nitrogen atom of asparagine), and 2 O-linked oligosaccharide chains (attached to the oxygen atom of serine or threonine). The sugar moiety directly attached to the fetuin polypeptide is usually a N-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a N-acetylneuraminic acid (NANA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from fetuin by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. Fetuin is also a carrier protein for growth factors. Fetuin is sometimes referred to as α2-HS-glycoprotein. Thus, it is considered that fetuin's biological effects on cultured cells are related to its carrier function for molecules with growth-promoting properties.

The synthesis of human α2-HS-glycoprotein is down-regulated by cytokines (hIL-1β, hIL-6) (Lebreton et al., J Clin. Invest. 64:1118-1129, 1979). Human fetuin levels are decreased (25-50%) in trauma patients (van Oss et al., J Trauma 15:451, 1975). Therefore, there is a need in the art to find a utility for fetuin and to understand fetuin's physiological role and the importance of its many negatively charged (at physiologic pH) sialic acid residues.

SUMMARY OF THE INVENTION

The present invention provides a method for helping to prevent miscarriages and pre-term labor during pregnancy, comprising administering an effective amount of a fetuin polypeptide. Preferably, the human fetuin polypeptide has a primary sequence according to SEQ ID NO. 1 or SEQ ID NO. 2 or a shortened fragment thereof having at least 250 amino acid residues.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the suppression of TNF secretion by spermine in the presence of fetuin (FIG.1A) or fetuin-specific polyclonal antibodies (FIG. 1B). HuPBMCs or RAW 264.7 cells were stimulated with E. coli endotoxin (LPS, 100 ng/ml) in the presence of spermine, human fetuin (α2-HS-glycoprotein), or polyclonal antibodies against fetuin. TNF levels in supernatants four hours post-LPS stimulation were determined by ELISA as previously described (Zhang et al., J Exp. Med. 185:1759-1768, 1997). Note that fetuin increases the TNF-suppressing activity of spermine, and anti-fetuin renders normal LPS-stimulated macrophages refractory to this suppression. A Student's t-test was performed and a P<0.05 was considered significantly different (*).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the new discovery that a fetal plasma glycoprotein, fetuin, is required for the inhibition of TNF production by spermine. Although fetuin was first described more than fifty years ago in fetal bovine serum, and subsequently found to share high homology to human fetuin (α2-HS-glycoprotein), its role in pregnancy and fetal development, until now, has been unknown. While investigating the mechanism underlying spermine-mediated suppression of TNF production in the murine macrophage-like cell line, RAW 264.7, we came upon the surprising discovery that macrophages lost their responsivity to spermine when cultured under low serum conditions. That is, despite the addition of cytokine-suppressing concentrations of spermine to these cells, the production of TNF was uninhibited by spermine after LPS stimulation.

It has previously been proposed that fetuin can function as a carrier of cell-modulating agents. We next showed that fetuin binds spermine by measuring the concentration of spermine after fractionation of a fetuin/spermine mixture (0-20 μM fetuin/100 μM spermine) via ultrafiltration. These results revealed that one molecule of fetuin is capable of binding 4-6 molecules of spermine. Since spermine and fetuin levels are both extremely high in the fetus and amnion, it now appears that they are ideally poised to counter-regulate TNF production in pregnancy.

EXAMPLE 1

This example illustrates the identification of fetuin as the protein responsible for some of the spermine-based activity observed in macrophage cultures. We added fractionated proteins from normal cells and assayed for their ability to restore the spermine-dependent inhibition of TNF production under serum-free culture conditions, because we hypothesized that these "spermine-non-responsive cells" had become deprived of a protein that was required to inhibit the production of TNF. After anion-exchange chromatography and SDS-PAGE gel elution, we isolated a single protein that mediated the responsivity of macrophage cultures to spermine. Computer-based protein database analysis of the N-terminal amino acid sequence identified this protein as fetuin.

The role of fetuin as a mediator of spermine inhibition of TNF production was confirmed by adding highly purified fetuin (Sigma, St. Louis, Mo.), together with spermine, to LPS-stimulated human peripheral blood mononuclear cells (HuPBMCs). As shown in FIG 1A, the level of TNF produced by LPS-stimulated HuPBMCs was significantly reduced by increasing the concentrations of fetuin for a given dose of spermine. Fetuin alone had no effect on TNF production (data not shown), indicating that both spermine and fetuin were required for the suppression of TNF synthesis.

We prepared polyclonal antiserum against purified fetuin, using standard techniques. The anti-fetuin polyclonal antibodies abrogated spermine-mediated suppression of TNF production from LPS-stimulated macrophages, whereas the control (pre-immune) serum did not (FIG. 1B). These data show that fetuin is required for spermine to suppress TNF production in normal human monocytes.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 2                                              - (2) INFORMATION FOR SEQ ID NO: 1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 359 amino                                                          (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -    (iii) HYPOTHETICAL: no                                                    -     (iv) ANTI-SENSE: no                                                      -      (v) FRAGMENT TYPE: N-terminal fragment                                  -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: human                                                  #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Ser Phe Val Leu Leu Phe Cys Leu Al - #a Gln Leu Trp Gly              #                 15                                                           - Cys His Ser Ile Pro Leu Asp Pro Val Ala Gl - #y Tyr Lys Glu Pro              #                30                                                            - Ala Cys Asp Asp Pro Asp Thr Glu Gln Ala Al - #a Leu Ala Ala Val              #                45                                                            - Asp Tyr Ile Asn Lys His Leu Pro Arg Gly Ty - #r Lys His Thr Leu              #                60                                                            - Asn Gln Ile Asp Ser Val Lys Val Trp Pro Ar - #g Arg Pro Thr Gly              #                75                                                            - Glu Val Tyr Asp Ile Glu Ile Asp Thr Leu Gl - #u Thr Thr Cys His              #                90                                                            - Val Leu Asp Pro Thr Pro Leu Ala Asn Cys Se - #r Val Arg Gln Gln              #                105                                                           - Thr Gln His Ala Val Glu Gly Asp Cys Asp Il - #e His Val Leu Lys              #               120                                                            - Gln Asp Gly Gln Phe Ser Val Leu Phe Thr Ly - #s Cys Asp Ser Ser              #               135                                                            - Pro Asp Ser Ala Glu Asp Val Arg Lys Leu Cy - #s Pro Asp Cys Pro              #               150                                                            - Leu Leu Ala Pro Leu Asn Asp Ser Arg Val Va - #l His Ala Val Glu              #               165                                                            - Val Ala Leu Ala Thr Phe Asn Ala Glu Ser As - #n Gly Ser Tyr Leu              #               180                                                            - Gln Leu Val Glu Ile Ser Arg Ala Gln Phe Va - #l Pro Leu Pro Val              #               195                                                            - Ser Val Ser Val Glu Phe Ala Val Ala Ala Th - #r Asp Cys Ile Ala              #               210                                                            - Lys Glu Val Val Asp Pro Thr Lys Cys Asn Le - #u Leu Ala Glu Lys              #               225                                                            - Gln Tyr Gly Phe Cys Lys Gly Ser Val Ile Gl - #n Lys Ala Leu Gly              #               240                                                            - Gly Glu Asp Val Arg Val Thr Cys Thr Leu Ph - #e Gln Thr Gln Pro              #               255                                                            - Val Ile Pro Gln Pro Gln Pro Asp Gly Ala Gl - #u Ala Glu Ala Pro              #               270                                                            - Ser Ala Val Pro Asp Ala Ala Gly Pro Thr Pr - #o Ser Ala Ala Gly              #               285                                                            - Pro Pro Val Ala Ser Val Val Val Gly Pro Se - #r Val Val Ala Val              #               300                                                            - Pro Leu Pro Leu His Arg Ala His Tyr Asp Le - #u Arg His Thr Phe              #               315                                                            - Ser Gly Val Ala Ser Val Glu Ser Ser Ser Gl - #y Glu Ala Phe His              #               330                                                            - Val Gly Lys Thr Pro Ile Val Gly Gln Pro Se - #r Ile Pro Gly Gly              #               345                                                            - Pro Val Arg Leu Cys Pro Gly Arg Ile Arg Ty - #r Phe Lys Ile                  #           359 355                                                            - (2) INFORMATION FOR SEQ ID NO: 2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 367 amino                                                          (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -    (iii) HYPOTHETICAL: no                                                    -     (iv) ANTI-SENSE: no                                                      -      (v) FRAGMENT TYPE: N-terminal fragment                                  -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: human                                                  #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - Met Lys Ser Leu Val Leu Leu Leu Cys Leu Al - #a Gln Leu Trp Gly              #15                                                                            - Cys His Ser Ala Pro His Gly Pro Gly Leu Il - #e Tyr Arg Gln Pro              #                 30                                                           - Asn Cys Asp Asp Pro Glu Thr Glu Glu Ala Al - #a Leu Val Ala Ile              #                 45                                                           - Asp Tyr Ile Asn Gln Asn Leu Pro Trp Gly Ty - #r Lys His Thr Leu              #                 60                                                           - Asn Gln Ile Asp Glu Val Lys Val Trp Pro Gl - #n Gln Pro Ser Gly              #                 75                                                           - Glu Leu Phe Glu Ile Glu Ile Asp Thr Leu Gl - #u Thr Thr Cys His              #                 90                                                           - Val Leu Asp Pro Thr Pro Val Ala Arg Cys Se - #r Val Arg Gln Leu              #                105                                                           - Lys Glu His Ala Val Glu Gly Asp Cys Asp Ph - #e Gln Leu Leu Lys              #               120                                                            - Leu Asp Gly Lys Phe Ser Val Val Tyr Ala Ly - #s Cys Asp Ser Ser              #               135                                                            - Pro Asp Ser Ala Glu Asp Val Arg Lys Val Cy - #s Gln Asp Cys Pro              #               150                                                            - Leu Leu Ala Pro Leu Asn Asp Thr Arg Val Va - #l His Ala Ala Lys              #               165                                                            - Ala Ala Leu Ala Ala Phe Asn Ala Gln Asn As - #n Gly Ser Asn Phe              #               180                                                            - Gln Leu Glu Glu Ile Ser Arg Ala Gln Leu Va - #l Pro Leu Pro Pro              #               195                                                            - Ser Thr Tyr Val Glu Phe Thr Val Ser Gly Th - #r Asp Cys Val Ala              #               210                                                            - Lys Glu Ala Thr Glu Ala Ala Lys Cys Asn Le - #u Leu Ala Glu Lys              #               225                                                            - Gln Tyr Gly Phe Cys Lys Ala Thr Leu Ser Gl - #u Lys Leu Gly Gly              #               240                                                            - Ala Glu Val Ala Val Thr Cys Thr Val Phe Gl - #n Thr Gln Pro Val              #               255                                                            - Thr Ser Gln Pro Gln Pro Glu Gly Ala Asn Gl - #u Ala Val Pro Thr              #               270                                                            - Pro Val Val Asp Pro Asp Ala Pro Pro Ser Pr - #o Pro Leu Gly Ala              #               285                                                            - Pro Gly Leu Pro Pro Ala Gly Ser Pro Pro As - #p Ser His Val Leu              #               300                                                            - Leu Ala Ala Pro Pro Gly His Gln Leu His Ar - #g Ala His Tyr Asp              #               315                                                            - Leu Arg His Thr Phe Met Gly Val Val Ser Le - #u Gly Ser Pro Ser              #               330                                                            - Gly Glu Val Ser His Pro Arg Lys Thr Arg Th - #r Val Val Gln Pro              #               345                                                            - Ser Val Gly Ala Ala Ala Gly Pro Val Val Pr - #o Pro Cys Pro Gly              #               360                                                            - Arg Ile Arg His Phe Lys Val                                                  #   367         365                                                            __________________________________________________________________________ 

We claim:
 1. A method for inhibiting pro-inflammatory cytokine production under conditions where extracellular spermine levels are elevated, comprising administering an effective amount of a human fetuin polypeptide.
 2. The method of claim 1 wherein the human fetuin polypeptide has a primary sequence according to SEQ ID NO. 1 or SEQ ID NO. 2, or a shortened fragment thereof having at least 250 amino acid residues. 